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High cell density perfusion process development for antibody producing Chinese Hamster Ovary cells
KTH, School of Biotechnology (BIO), Industrial Biotechnology. (Animal Cell Technology)ORCID iD: 0000-0002-0841-8845
2017 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Perfusion operation mode is currently under fast expansion in mammalian cell based manufacturing of biopharmaceuticals, not only for labile drug protein but also for stable proteins such as monoclonal antibodies (mAbs). Perfusion mode can advantageously offer a stable cell environment, long-term production with high productivity and consistent product quality. Intensified high cell density culture (HCDC) is certainly one of the most attractive features of a perfusion process due to the high volumetric productivity in a small footprint that it can provide. Advancements in single-use technology have alleviated the intrinsic complexity of perfusion processes while the maturing in cell retention devices has improved process robustness. The knowledge for perfusion process has been gradually built and the “continuous” concept is getting more and more acceptance in the field.

This thesis presents the development of robust perfusion process at very high cell densities in various culture systems. Four HCDC perfusion systems were developed with industrial collaborators with three different mAb producing Chinese Hamster Ovary (CHO) cell lines: 1-2) WAVE Bioreactor™ Cellbag prototype equipped with cell separation by hollow fiber filter utilizing Alternating Tangential Flow (ATF) and Tangential Flow Filtration (TFF) techniques; 3) Fiber matrix based CellTank™ prototype; 4) Glass stirred tank bioreactor equipped with ATF. In all the systems, extremely high viable cell densities above 130 million viable cells per milliliter (MVC/mL) up to 214 MVC/mL were achieved. Steady states were maintained and studied at 20-30 MVC/mL and 100-130 MVC/mL for process development. Perfusion rate selection based on cell specific perfusion rate (CSPR) was systematically investigated and exometabolome study was performed to explore the metabolic footprint of HCDC perfusion process.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2017. , p. 79
Series
TRITA-BIO-Report, ISSN 1654-2312 ; 2017:14
Keywords [en]
WAVE Bioreactor, Cellbag, ATF, TFF, hollow fiber, CSPR, CellTank, SUB, single-use-bioreactor, disposable, perfusion rate
National Category
Bioprocess Technology
Research subject
Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-207600ISBN: 978-91-7729-450-4 (print)OAI: oai:DiVA.org:kth-207600DiVA, id: diva2:1097269
Public defence
2017-06-14, FB54, Albanova, plan_5, Stockholm, 14:00 (English)
Opponent
Supervisors
Note

QC 20170523

Available from: 2017-05-23 Created: 2017-05-22 Last updated: 2017-05-29Bibliographically approved
List of papers
1. Very high density of CHO cells in perfusion by ATF or TFF in WAVE bioreactor. Part I. Effect of the cell density on the process
Open this publication in new window or tab >>Very high density of CHO cells in perfusion by ATF or TFF in WAVE bioreactor. Part I. Effect of the cell density on the process
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2013 (English)In: Biotechnology progress (Print), ISSN 8756-7938, E-ISSN 1520-6033, Vol. 29, no 3, p. 754-767Article in journal (Refereed) Published
Abstract [en]

High cell density perfusion process of antibody producing CHO cells was developed in disposable WAVE Bioreactor using external hollow fiber filter as cell separation device. Both classical tangential flow filtration (TFF) and alternating tangential flow system (ATF) equipment were used and compared. Consistency of both TFF- and ATF-based cultures was shown at 20-35 x 106 cells/mL density stabilized by cell bleeds. To minimize the nutrients deprivation and by-product accumulation, a perfusion rate correlated to the cell density was applied. The cells were maintained by cell bleeds at density 0.9-1.3 x 108 cells/mL in growing state and at high viability for more than 2 weeks. Finally, with the present settings, maximal cell densities of 2.14 x 108 cells/mL, achieved for the first time in a wave-induced bioreactor, and 1.32 x 108 cells/mL were reached using TFF and ATF systems, respectively. Using TFF, the cell density was limited by the membrane capacity for the encountered high viscosity and by the pCO2 level. Using ATF, the cell density was limited by the vacuum capacity failing to pull the highly viscous fluid. Thus, the TFF system allowed reaching higher cell densities. The TFF inlet pressure was highly correlated to the viscosity leading to the development of a model of this pressure, which is a useful tool for hollow fiber design of TFF and ATF. At very high cell density, the viscosity introduced physical limitations. This led us to recommend cell densities under 1.46 x 108 cell/mL based on the analysis of the theoretical distance between the cells for the present cell line.

Place, publisher, year, edition, pages
Wiley-Blackwell, 2013
Keywords
wave bioreactor, alternating tangential flow hollow fiber, tangential flow filtration hollow fiber, perfusion, cell broth viscosity, Chinese Hamster Ovary cell
National Category
Biological Sciences
Identifiers
urn:nbn:se:kth:diva-124990 (URN)10.1002/btpr.1704 (DOI)000320387300019 ()2-s2.0-84879258603 (Scopus ID)
Note

QC 20130806

Available from: 2013-08-06 Created: 2013-08-02 Last updated: 2017-05-23Bibliographically approved
2. Very high cell density perfusion of CHO cells anchored in a non-woven matrix-based bioreactor
Open this publication in new window or tab >>Very high cell density perfusion of CHO cells anchored in a non-woven matrix-based bioreactor
2015 (English)In: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 213, p. 28-41Article in journal (Refereed) Published
Abstract [en]

Recombinant Chinese Hamster Ovary (CHO) cells producing IgG monoclonal antibody were cultivated in a novel perfusion culture system CellTank, integrating the bioreactor and the cell retention function. In this system, the cells were harbored in a non-woven polyester matrix perfused by the culture medium and immersed in a reservoir. Although adapted to suspension, the CHO cells stayed entrapped in the matrix. The cell-free medium was efficiently circulated from the reservoir into- and through the matrix by a centrifugal pump placed at the bottom of the bioreactor resulting in highly homogenous concentrations of the nutrients and metabolites in the whole system as confirmed by measurements from different sampling locations. A real-time biomass sensor using the dielectric properties of living cells was used to measure the cell density. The performances of the CellTank were studied in three perfusion runs. A very high cell density measured as 200 pF/cm (where 1 pF/cm is equivalent to 1 x 106 viable cells/mL) was achieved at a perfusion rate of 10 reactor volumes per day (RV/day) in the first run. In the second run, the effect of cell growth arrest by hypothermia at temperatures lowered gradually from 37 C to 29 C was studied during 13 days at cell densities above 100 pF/cm. Finally a production run was performed at high cell densities, where a temperature shift to 31 C was applied at cell density 100 pF/cm during a production period of 14 days in minimized feeding conditions. The IgG concentrations were comparable in the matrix and in the harvest line in all the runs, indicating no retention of the product of interest. The cell specific productivity was comparable or higher than in Erlenmeyer flask batch culture. During the production run, the final harvested IgG production was 35 times higher in the CellTank compared to a repeated batch culture in the same vessel volume during the same time period.

Place, publisher, year, edition, pages
[Zhang, Ye; Chotteau, Veronique] KTH, Royal Inst Technol, Cell Technol Grp CETEG, Sch Biotechnol,Dept Ind Biotechnol Bioproc Design, SE-10691 Stockholm, Sweden. [Stobbe, Per] PerfuseCell, DK-2840 Holte, Denmark. [Silvander, Christian Orrego] Belach Biotekn, SE-14250 Skogas, Sweden.: , 2015
Keywords
Disposable bioreactor, On-line biomass sensor, IgG production, Dielectric spectroscopy, Hypothermia
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:kth:diva-176326 (URN)10.1016/j.jbiotec.2015.07.006 (DOI)000362286400005 ()26211737 (PubMedID)2-s2.0-84942837005 (Scopus ID)
Note

QC 20151117

Available from: 2015-11-17 Created: 2015-11-03 Last updated: 2017-05-23Bibliographically approved
3. Optimization of the cell specific perfusion rate in high cell density perfusion process
Open this publication in new window or tab >>Optimization of the cell specific perfusion rate in high cell density perfusion process
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(English)Manuscript (preprint) (Other academic)
National Category
Bioprocess Technology
Identifiers
urn:nbn:se:kth:diva-207741 (URN)
Note

QC 20170523

Available from: 2017-05-23 Created: 2017-05-23 Last updated: 2017-05-23Bibliographically approved
4. Metabolic footprinting of CHO cell culture bioprocess data in fed-batch and perfusion mode using LC-MS data and multivariate analysis
Open this publication in new window or tab >>Metabolic footprinting of CHO cell culture bioprocess data in fed-batch and perfusion mode using LC-MS data and multivariate analysis
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(English)Manuscript (preprint) (Other academic)
National Category
Bioprocess Technology
Identifiers
urn:nbn:se:kth:diva-207742 (URN)
Note

QC 20170523

Available from: 2017-05-23 Created: 2017-05-23 Last updated: 2017-05-23Bibliographically approved

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